Facile Imaging of Murine Bone and Soft Tissue Using the Kodak Digital X-Ray Specimen System
Sean Orton,a Ryan Park,b M. Catherine Muenker,a Manisha Patel,a Hitenkumar Patel,b Samuel Li,b Rao V. L. Papineni,a Douglas Vizard,a Gilbert Feke,a Grant Dagliyan,b Peter S. Conti,b Edward G. Grant,b William McLaughlin,a W. Matthew Leevy,a,c †
a Carestream Molecular Imaging, a division of Carestream Health, Inc., 4 Science Park, New Haven, CT 06511
b Molecular Imaging Center, University of Southern California, 2250 Alcazar Street, CSC/IGM 103, Los Angeles, CA 90033
c Department of Chemistry and Biochemistry, 251 Nieuwland Science Hall, University of Notre Dame, Notre Dame, IN 46556
†Correspondingauthor contact information:
Phone: 203-786-5652. E-mail: warren.leevy@carestreamhealth.com;
Phone: 203-786-5645. E-mail: William.mclaughlin@carestreamhealth.com
INTRODUCTION:
Planar X-ray imaging has been utilized for decades both in the clinic, and also in the laboratory for specimen radiography of various biological samples. This imaging has traditionally been accomplished in both settings using radiographic film, which can be developed to yield an X-ray image. While film-based technology is still in use today, methods for digital capture of X-ray images have been advanced over the last two decades, and have emerged as the standard technology for clinical use today. However, digital X-ray imaging for specimen radiography of small animals has lagged the rapid adaptation noted in the clinics. The resolution levels applied to human radiology are insufficient to produce satisfactory images for use with small laboratory animals. Recent developments in planar X-ray equipment from Carestream Molecular Imaging (Formerly Kodak Molecular Imaging) have engendered advances in resolution to image small animals with digital technology.
One instrument that has emerged with leading-edge technology for pre-clinical research of biological specimens is the Kodak Digital X-ray Specimen System (DXS). This instrument utilizes a unique setup compared to traditional digital imaging systems. It incorporates a cooled, 4-megapixel charge coupled device (CCD) camera that is focused onto a sensitive phosphor screen. When this screen is irradiated from above with low energy X-rays (35keV), the phosphor transduces the radiation to visible light that is captured by the CCD camera. During specimen radiography, the resolution of the Kodak DXS surpasses 25 line pairs per millimeter (lp/mm). By comparison, other instruments that use the more common digital X-ray sensing pads usually typically claim resolution of around 10 lp/mm. Indeed, the extra resolution afforded by the Kodak DXS is critical when performing experiments on small animals in which fine structural detail must be observed. Here we show the application of this instrument to bone and soft tissue imaging in mice.
RESULTS:
Several anatomical features may be noted in an animal by observing its innate contrast during X-ray imaging. Figure 1 shows a typical X-ray image of a mouse obtained using a Kodak DXS instrument (see Experimental section for methods). This 16-bit image was acquired in 180 seconds at a 120 mm field of view (see experimental section for details). It is displayed in the greyscale color scheme, with intensity and spatial scale bars located in the upper and lower right corners, respectively. For the purposes of this application note, the intensity signal orientation has been inverted such that darker areas on the animal have both increased
contrast and pixel intensity values. First, the classic feature studied using X-ray imaging is the bone framework, noted by Figure 1A. Since bones are dense structures they absorb X-rays and appear dark. One can immediately note the fine structure in bones that are sub-millimeter in scale, such as the rib cage and fibula bone in each leg. Many disease models involving the skeleton may be non-invasively studied using such images. These include bone growth and damage in response to environmental or physical inputs. Furthermore, subtle changes in bone density may also be measured using the Kodak DXS software package.
While bones are dense and provide positive contrast that appears black, air is obviously of low density and absorbs very little X-ray radiation. Thus, locations in an animal that contain gases give contrast toward the white end of the intensity spectrum shown in Figure 1. One area in which air is abundant is in the lungs (Figure 1 C). In fact, the rib cage is often considered a cavern of air due to the presence of the lungs, which appear as a triangular pattern on each half of the rib cage. Another area in which gas tends to build up and give negative contrast on X-ray is in the bowels (Figure 1F). Nevertheless, the air cavern of the rib cage is home to another important organ: the heart (Figure 1B). Since the tissue comprising the heart has higher density than air, it gives positive (dark) contrast in comparison to the lungs. Indeed, since this organ is effectively surrounded by air from the lungs, it is often described as a “heart shadow” in the rib cage.
Two disease states in the rib cage area may be readily imaged using X-ray. The first is known as pulmonary edema. In this case, the lungs begin accumulating fluid, and their air capacity is drastically reduced. This results in a loss of negative contrast of the lungs in the rib cage that is readily observable by X-ray. Syková and colleagues recently published a study in which the Kodak Digital X-ray Specimen System was used to image pulmonary edema in rats that was induced by spinal injury.1 A second physiological measurement that is accessible in the rib cage is the size of the heart shadow as it expands into the lung area. If its size increases too much, it is an indication of pericardium swelling, a condition in which the region about the heart fills with liquid. While the heart and lungs are soft tissue systems that are readily observed by X-ray, other organs possess little innate contrast to permit their imaging and require an alternate strategy.
Organs like the stomach, intestines, kidneys, bladder and liver give limited contrast during X-ray imaging. Figure 1D-F shows the lack of discernable contrast from each of these various tissue systems in the abdomen. However, through the use of contrast agents, these organs can be delineated and studied in a non-invasive fashion using the X-ray modality. Molecules that incorporate atoms with exceptional X-ray absorption properties are typically utilized as contrast agents. Barium and iodine meet this criterion and are two of the most
widely used atomic components of X-ray contrast agents. Indeed, both of these reagents have been utilized in the clinic for decades to perform gastric and heart perfusion imaging, among others. Here we provide examples in a pre-clinical research setting in which these reagents delineate four different organs of living mice.
Barium is an X-ray contrast agent that is commonly used in the clinic for imaging of the gastrointestinal tract. Since barium salts are generally insoluble and inert, they may be safely used in the human GI tract, either orally or rectally, to provide contrast during X-ray imaging. After imaging is complete, the barium is excreted from the subject without absorption into the body through the intestines. Many products incorporating barium are available today for human consumption including milkshakes, and other solutions used for enema-based applications. We have adapted this approach for use with small animals. Figure 2 shows a mouse one hour after consuming 40 mg of a 1:1 mixture of barium sulfate and creamy peanut butter. Frame A presents the animal in the prone (ventral) position, and subsequent frames show the animal on its right side (B), its back, or dorsal side (C), and on its left side (D). The GI tract in the abdomen is clearly delineated by the contrast agent. Frames A and C show the path of the intestines as they descend through the abdomen. Meanwhile, frames B and D provide depth information, demonstrating that the GI tract is primarily located within the first 1 cm of depth in the abdomen. In addition, the stomach is also noted in frames B, C, and D as a “U” shaped loop located just underneath the rib cage of the animal. The stomach is more centrally located in the animal as noted from viewing the animal at different angles. Barium assisted gastric imaging gave an average target/background (T/BKG) ratio of 1.6 +/- 0.2 for the stomach and 2.00 +/- 0.26 for the intestines (n = 3, standard error of the mean (SEM) reported in all cases). Taken together, this technique provides a rapid and effective means to identify both the stomach and intestines by X-ray imaging aided with a barium contrast agent. While agents like barium are helpful for imaging the GI tract, their insolubility limits their application in the blood stream and its perfused tissues.
Iodine is a synthetically accessible atom with sufficient electron density to yield X-ray contrast. Thus, it has been incorporated into several compounds that may be synthesized as water soluble through contrast agents. These reagents are generally used for the purposes of intravenous injection since they are not harmful, and will be excreted from the subject via the renal pathway. Several iodine based contrast agents are commercially available for use in humans for imaging of heart vasculature and other bulk tissues. We have utilized one such agent, known as Visipaque®, to image the renal system of mice. Figure 3A shows a mouse before introduction of any contrast agent. Figure 3B shows the animal 5 min. after intravenous injection of 200 µL of Visipaque® (320 mg/ml iodine). After this short time period, the contrast agent began concentrating in the kidneys, thus providing clear contrast in them. Figure 3C shows the animal after 45 minutes, in which the kidneys are still contrasted
as they continue to clear the contrast agent, and the bladder also appears as the animal excretes the water-soluble agent. At the five minute time point, Visipaque® gave average T/BKG ratios of 1.52 +/- 0.1 for the inner core, or medulla of the kidney, while the outer portion, or cortex, had values of 1.24 +/- 0.06. At the 45-minute time point, the bladder had a T/BKG value of 2.06 +/- 0.13. These X-ray images demonstrate that the bulk physiology of the kidneys and bladder may be observed by X-ray. In addition, the kinetics of perfusion of the contrast agent may also provide another metric with which to measure kidney health.
CONCLUSIONS:
The X-ray modality provides an effective means with which to non-invasively image both bone and soft tissue in living animals. Indeed, many diseases are marked by anatomical changes in the bulk physiology of the specimen under study. Recent advances in X-ray technology provided by the Kodak DXS System have made possible the digital imaging of these bulk changes. The innate X-ray contrast of bones, lungs and heart permits their imaging using the Kodak DXS System. In the case of the kidneys, bladder, stomach and intestines, X-ray contrast agents incorporating barium or iodine may be readily utilized to visualize these organs. In both cases, digital imaging makes the process both rapid and facile. Indeed, the advantages conveyed by digital imaging as opposed to film imaging will accelerate the pace of research of disease models as the technology becomes widely adapted for pre-clinical studies.
EXPERIMENTAL:
Digital X-Ray Imaging. In each case, the animals were anesthetized using isofluorane inhalation (3%) from an exhaust manifold within the image station. The animals were then imaged in X-ray mode using the following settings: Binning = 1 x 1, F-stop = 2.8, Aluminum Filter = 0.4 mm, Acquisition time = 180 sec., Field of View = 120 mm. The animal received an approximate radiation dose of 1.4 Rad during imaging, which is significantly below the generally accepted safety threshold of 100 Rad.
1 Šedý, J.; Urdzíková, L.; Likavčanová, K.; Hejčl, A.; Jendelová, P.; and Syková, E. NeuroSci. Lett. 2007, 423, 167-171
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